Bacteriophage PRD1 as a nanoscaffold for drug loading

Viruses are very attractive biomaterials owing to their capability as nanocarriers of genetic material. Efforts have been made to functionalize self-assembling viral protein capsids on their exterior or interior to selectively take up different payloads. PRD1 is a double-stranded DNA bacteriophage comprising an icosahedral protein outer capsid and an inner lipidic vesicle. Here, we report the three-dimensional structure of PRD1 in complex with the antipsychotic drug chlorpromazine (CPZ) by cryo-electron microscopy. We show that the jellyrolls of the viral major capsid protein P3, protruding outwards from the capsid shell, serve as scaffolds for loading heterocyclic CPZ molecules. Additional X-ray studies and molecular dynamics simulations show the binding modes and organization of CPZ molecules when complexed with P3 only and onto the virion surface. Collectively, we provide a proof of concept for the possible use of the lattice-like organisation and the quasi-symmetric morphology of virus capsomers for loading heterocyclic drugs with defined properties.

Due to significant aggregation of PRD1 particles in the presence of CPZ, sample and grid optimization were essential steps prior to data collection for high-resolution 3D reconstruction (sample concentration: ~1 mg/ml of PRD1 and ~60 mM of CPZ). Movies were recorded at the OPIC facility at the Division of Structural Biology at Oxford University-WHG (UK) on a Titan Krios microscope (ThermoFisher FEI) with a Falcon III camera in linear mode and a nominal magnification of 59,000 X, calibrated to 1.39 Å/pix (Table  S1 †).
Image processing, 3D reconstruction and model refinement Movie pre-processing was performed using MotionCorr2.1 42 , ctf-corrected with CtfFind4.2 43 . All subsequent processing was performed using RELION 2.0 and RELION 3.0 44 . Owing to the high density and substantial overlap of virions conventional software failed to correctly distinguish an adequate number of particles; particles were instead manually selected using the RELION 2.0 interface. Extracted virions were then subjected to 2D and then 3D classification with icosahedral symmetry imposed and using a reference volume derived from the published PRD1 structure (PDB ID 1w8x) 13 . After a 3D auto-refine job, three rounds of CTF refinement were then performed using RELION 3.0. This resulted in a final post-processed electron density map exhibiting a 3.9 Å resolution as estimated by the gold-standard FSC (0.143 threshold) within a threshold mask (10 Å low-pass filter, 0.02 initial binarisation threshold, 5 pixels extension of the binary map plus 5 soft-edge pixels) (Fig. S2 †). The P3 MCPs making up an icosahedral asymmetric unit (PDB ID 1w8x) were fitted into the corresponding density using CHIMERA 39 . The density external to the capsid not accounted for by the P3 capsomers was interpreted as molecules of CPZ and modelled by MD simulations (see below).
Crystallization of P3 in complex with CPZ, data collection, and structure determination and refinement Purified P3 protein was buffer exchanged into 10 mM Tris-HCl pH 8.5, 300 mM NaCl and concentrated to 5 mg/mL using a 10 kDa cut off Amicon concentrator device. Plates were then set up using the P3 trimer alone using previously described conditions 23 . Soaks were performed with 1 mM, 15 mM and 25 mM final concentrations of CPZ for 30 minutes. Diffraction data were collected at room temperature at beamline I24, Diamond Light Source (UK) using a 50 x 50 μm beam size, at either 50 % (native), or 100 % (soaked) transmission, with 0.1 o oscillation between frames, 0.01-0.05 s exposure time as a 360o wedge. Data were indexed, integrated, scaled and merged using the xia2-dials pipeline (Table S2 †). As determined previously, all crystals were indexed with a P2 1 2 1 2 1 space group. Intensities were converted to structure factor amplitudes using TRUNCATE 45 , before model refinement using PHENIX 46 . A round of rigid body refinement with the P3 trimer (PDB ID 1hx6) as initial model was performed prior positional and B-factor refinements with torsion-NCS restraints; the latter were relaxed in the last three refinement cycles (Table S2 †).

Virus infectivity and thermal stability assays
Purified PRD1 particles (0.5 mg/ml) were treated with 30 or 60 mM CPZ in 20 mM potassium phosphate pH 7.2 or pH 6.0, 1 mM MgCl 2 for 15 min or 2 hrs at room temperature. Non CPZ-treated particles were used as a control. CPZ-treated PRD1 (30 mM CPZ) and control particles were either left in the buffer above or in the same buffer further supplemented with 150 mM or 300 mM NaCl (incubated for 30 min at 18 C). Sedimentation of the particles was analysed by rate zonal centrifugation in 5-20% (w/v) sucrose gradient (20 mM potassium phosphate pH 6.0, 1 mM MgCl 2 ; Sorvall TH641 rotor, 24 000 rpm, 50 min, +15 o C). Specific infectivities of the light-scattering bands were calculated based on plaque assay data and absorbance measurements (NanoDrop). Protein composition was analysed by protein gel electrophoresis (16% w/v acrylamide) and Coomassie blue staining 47 . Solutions of PRD1 with or without 60 mM CPZ (CPZ solution prepared as above) at either pH 6.0 or pH 7.2 as well as CPZ alone at these two probe pHs were prepared and incubated at room temperature for 15 minutes before being put on ice. No discolouration, nor evidence of protein precipitation was observed for any of the samples. Samples were then aliquoted into a 96-well PCR plate containing SYBR green DNA dye (Thermo Fisher Scientific) in triplicate, sealed and gently centrifuged for 3 min at 4 o C. The plate was then heated in a Mx3005p qPCR machine (Agilent Technologies, USA) from 25 to 97 o C in 1 o C min −1 increments for 30 s, with temperature cycles following an expanding saw-tooth profile, where fluorescence changes were monitored with exci/em 492/517 nm at the 25 o C 'troughs' of this saw-tooth profile 48 .

Quantum mechanical calculations and molecular dynamic simulations of CPZ
For quantum mechanical calculations, full geometry optimizations of CPZ monomers, dimers and trimers excluding the N,Ndimethylpropanamine moiety tail were carried out with Gaussian 16 using the M06-2X hybrid functional and a 6-31+G(d,p) basis set with ultrafine integration grids 49 . Bulk solvent effects in water were considered implicitly through the IEF-PCM polarizable continuum solvent model 50 . The possibility of different conformations was considered for all structures. All stationary points were characterized by a frequency analysis performed at the same level used in the geometry optimizations from which thermal corrections were obtained at 298.15 K. The quasi-harmonic approximation reported by Ribeiro et al 51 . was used to replace the harmonic oscillator approximation for the calculation of the vibrational contribution to enthalpy and entropy. All computed structures can be obtained from authors upon request. The QM-optimized geometries for CPZ monomers were used as starting coordinates for the Molecular Dynamics (MD) simulations of CPZ loading at the MCP P3 surface. Protein models were generated extracting 618 and 658 residues capsid fragments from the Please do not adjust margins Please do not adjust margins regions underlying the sphere-like and elongated density, respectively. For the protein model underlying the sphere-like density, only the V1 jellyroll of P3 was conserved (residues S35 to L240), for a total of three V1 jellyrolls belonging to different chains. For the model underlying the elongated density, a four-chains model was constructed containing two non-equivalent fragments of V1 and V2 jellyrolls corresponding to residues Y36 to Q239 and L257 to T381, respectively. Initial geometries for the CPZ-loaded capsid protein were generated by immersing protein models in a rectangular box of 3,200 CPZ molecules using the Packmol package 52 . Then, excess CPZ molecules were trimmed leaving an ellipsoidal (431 molecules) and a spherical (266 molecules) distribution to represent cigar-like and sphere-like densities, respectively. CPZ has a pKa of 8.6, meaning that its protonated (charged) and deprotonated (neutral) forms will coexist at pH 7.2 21 . As an approximation, the protonation state of CPZ was randomized to obtain an approximate 1:1 ratio of the two forms. MD simulations were carried out with the AMBER 20 package. The ff14SB and the general Amber force fields (GAFF2) were used to describe the protein and CPZ, respectively [53][54][55] . CPZ parameters were generated with the antechamber module of AMBER, using the GAFF2 force field and with partial charges set to fit the electrostatic potential generated with HF/6-31G(d) using the RESP method 56 . Charges were calculated according to the Merz-Singh-Kollman scheme using Gaussian 16 46 . Protein complexes were immersed in a water box with a 10 Å buffer of TIP3P water molecules and neutralized by adding explicit K + or Clcounterions 57 . A two-stage geometry optimization approach was performed. The first stage minimizes only the positions of solvent molecules and ions, and the second stage is an unrestrained minimization of all the atoms in the simulation cell. The systems were then heated by increasing the temperature from 0 to 300 K under a constant pressure of 1 atm and periodic boundary conditions. Harmonic restraints of 10 kcal/mol were applied to the solute, and the Andersen temperature coupling scheme was used to control and equalize the temperature 58,59 . The time step was kept at 1 fs during heating stages, allowing potential inhomogeneities to self-adjust. Water molecules were treated with the SHAKE algorithm such that the angle between the hydrogen atoms is kept fixed through the simulations 60 . Long-range electrostatic effects were modelled using the particle mesh Ewald method 61 . An 8 Å cut-off was applied to Lennard-Jones interactions. Each system was equilibrated for 2 ns with a 2 fs time step at a constant volume and temperature of 300 K. Production (400 ns) was run in the NVT ensemble imposing harmonic constraints on the alpha carbons of the protein models with the exception of the flexible loops in proximity to the CPZ aggregates The theoretical densities of CPZ molecules fitting the experimental sphere-like and cigar-like extra electronic densities were estimated from MD simulations. To this aim, a CPZ aggregate model consisting of 1000 CPZ molecules embedded in aperiodic box of water molecules was generated. The protonation state of each CPZ molecule was randomized to obtain a total 1:10 distribution of charged:neutral species. This system was subjected to MD simulation using the same protocol described above. Along the MD simulation, the CPZ aggregate assumes a spherical shape with charged CPZs lining the sphere's surface and neutral CPZs packed at the solvent-excluded volume. Once the system was equilibrated (i.e. when the cluster acquired a constant volume), different regions of the large CPZ aggregate was fitted into both extra-density isosurfaces (isovalues: sphere-like density = 0.0555 e-/ Å -3 , cigar-like density = 0.0473 e-/ Å -3 ) using the Fit in map tool as implemented in Chimera software 41 . To obtain an average number of CPZ molecules from the heterogeneous molecular distribution of our aggregate model, five snapshots extracted from the MD simulation corresponding to the last 5 ns of the 400 ns simulation were selected. For each snapshot, five randomly selected regions of the aggregate were fitted into the extra-density isosurfaces, affording a total of 25 independent fittings for each extra-density shape. Averaging over these 25 values, a mean of 2.99 CPZ molecules for the sphere-like density (standard deviation = 0.25) and 8.39 for the cigar-like density (standard deviation = 0.37) were obtained. These values were calculated assuming that complete molecules reside within the extra electronic densities. Translating these values into densities required an estimation of the volume enclosed in the extra-density isosurfaces (~1260 Å 3 for sphere-like and ~3800 Å 3 for cigar-like densities, respectively), for which the Measure Volume and Area tool in Chimera was used. Considering the molecular mass of neutral CPZ (318.86 g mol -1 ), theoretical densities ρ were derived for the two extra-density shapes. In both cases, we computed ρ for each number of CPZ molecules in each snapshot (25 independent fittings) using the following formula: where ρ is the density in g cm -3 , N A is Avogadro's number, MW is the molecular weight in g mol -1 and V is the volume of the extradensity region in cm 3 . The 25 density values were averaged and are presented with their standard deviation. Figure S4. Effect of ionic strength on PRD1-CPZ particles. (a) Comparison of 2D cryo-images of bacteriophage PRD1 in presence of 30 mM CPZ and 150 and 300 mM NaCl, respectively (NaCl was added after preparing PRD1+CPZ solution). Scale bar, 50 nm; (b) Light-scattering zones after NaCl addition to PRD1 sample with and without CPZ and after rate zonal centrifugation in a sucrose gradient (PRD1 is known to be stable at 300 mM 62 ).

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Please do not adjust margins Figure S6. Sphere-like extra density modelled by MD simulations. Top, evolution of the number of charged (in blue) and neutral (in red) CPZ molecules at the core of the CPZ aggregate along the MD simulation. The number of charged CPZ molecules decreases sharply, indicating that already at 180 ns the aggregate core is mainly composed of neutral CPZ molecules. Bottom, evolution of the number of water molecules and size of the CPZ aggregate along the MD simulation. The aggregate shrinks as water molecules are excluded from its increasingly hydrophobic core. Missing points in the 170-250 and 300-350 ns ranges correspond to asymmetric CPZ molecules distributions that could not be fitted to a spherical surface.

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Please do not adjust margins Figure S7. Cigar-like extra density modelled by MD simulations. Top, evolution of the number of charged and neutral CPZ molecules at the core of the CPZ aggregate along the MD simulation. The number of charged CPZ molecules decreases sharply, indicating that already at 180 ns the aggregate core is mainly composed of neutral CPZ molecules. Bottom, evolution of the number of water molecules and size of the CPZ aggregate along the MD simulation. The aggregate shrinks as water molecules are excluded from its increasingly hydrophobic core. Missing points in the 80-130 ns range correspond to asymmetric CPZ molecules distributions that could not be fitted to an elongated surface.

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Please do not adjust margins     Table S4. Sphere-like extra density Number of neutral and protonated CPZ molecules in the first solvation shell of selected residues of P3 after 400 ns of molecular dynamics. We observe more contacts with charged than with neutral CPZ molecules. This matches the observation that protonated CPZ molecules line the exterior of the aggregate and form attractive electrostatic interactions with negatively charged residues such as D and E.